The overall goal of this project is to develop methods for the production of functional platelets in vitro that could be used for clinical transfusion. Transfusion of allogeneic platelets is a critical part of supportive therapy for patients undergoing chemotherapy, stem cell transplantation, and those with thrombocytopenia due to increased consumption or inadequate production. However, the supply of transfusable platelets is dependent on volunteer donors. Furthermore, platelets have a short shelf-life and may be contaminated with bacterial or viral pathogens. Our proposal is based on the following hypotheses: 1) rational modulation of extrinsic and intrinsic factors will allow expansion of primary megakaryocytes in a manner that maintains an immature phenotype; 2) changes in culture conditions will facilitate terminal maturation and platelet shedding of the expanded MKs; and 3) platelet-like fragments, formed under these conditions, will have hemostatic activity, comparable to normal platelets. Specific Aims are: Aim 1: Develop methods for the expansion of megakaryocytes (MKs) from bone marrow or umbilical cord blood derived progenitors. Aim 2: Promote thrombopoiesis (i.e. fragmentation) of MKs and megakaryocyticcell lines in vitro. Aim 3: Test function of platelets produced in vitro. Successful completion of these Aims will lead directly to studies of in vitro derived platelets in non- human primates, with extensive testing to determine safety, efficacy, and platelet survival in vivo as well as immunogenicity upon repeated administration. This may eventually result in the production of autologous and/or donor-derived platelets from expanded progenitors in order to diminish alloimmunization, improve survival, and eliminate the risk of infectious complications.